In proteomic research, two complementary approaches (bottom-up and top-down) are commonly used for protein analysis. The bottom-up approach has progressed rapidly due to the advancement of modern mass spectrometry (MS), enabling identification and quantitation of hundreds or even thousands of proteins in single analysis. However, this approach can miss the information of post-transitional modification, mutations and proteolytic cleavage, and therefore other approaches are constantly sought. The top-down approach is a great alternative, in which intact proteins are individually analyzed. One of the most challenging tasks in the latter approach is to isolate proteins from a background of many other proteins and complex matrices. Often, this is beyond the resolving power of a one-dimensional separation technique. Vast effort has been invested on exploiting multi-dimensional separation strategies and progress has been made recently.
Two-dimensional electrophoresis (2DE) is a powerful method for separating intact proteins. It quickly became routine after its introduction in 1975. However, extracting individual proteins after 2DE separation is tedious and time-consuming. In the last decade or so, two-dimensional liquid chromatography (2D-LC), also referred to herein as two-dimensional high performance liquid chromatography (2D-HPLC), has received considerable attention due to its attractive features such as great number of chromatography mode choices, high resolving power, convenience for collecting resolved-proteins, and straightforwardness for automating 2D HPLC.
While some 2D HPLC techniques analyze only portions of the first dimension (first-D) effluents, a comprehensive 2D HPLC analyzes all of them, preventing losses of any proteins. One of the early comprehensive 2D HPLC systems was constructed by Bushey and Jorgenson (Bushey, M. M. & Jorgenson, J. W. Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography of proteins. Anal. Chem. 62, 161-167 (1990)). Through an eight-port valve, these authors coupled cation exchange and size exclusion chromatography for intact protein separations.
A major challenge in current 2D HPLC is the limited separation speed in the second dimension (second-D). Because current 2D HPLC systems employ one first-D column and one second-D column, the first-D effluent is fractionated and sequentially analyzed by the second-D. In order to retain the first-D resolution, one has to fractionate its effluent frequently (or in small segments) to minimize the re-mixing of the resolved analytes. Often, the effluent fractions must be parked somewhere for later or off-line analyses, resulting in a slow and tedious analysis of the sample. An improved 2D HPLC process which results in faster separation would be desirable. It is to this goal that the novel technology of the present disclosure is directed.